We propose to expand the scope of R01 CA57973-16 "Structure and function of the HCV RNA replicase", which currently funds our studies of hepatitis C virus (HCV) replication. HCV infects over 130 million people worldwide, with often devastating consequences including liver cirrhosis and hepatocellular carcinoma. We are currently investigating the structures and functions of individual viral proteins that make up the HCV RNA replication complex, we are also attempting to catalog interactions between viral proteins that contribute to the architecture of the replicase, and ultimately to reconstitute replication in vitro. In the one month since the renewal of R01 CA57973-16 funding, we have made excellent progress towards the aims of the parent grant, including preparing a manuscript describing the mechanism of NS3 helicase activity (Gu M and Rice CM, in preparation). We are now proposing to extend the aims of the parent grant to include the identification and characterization of functional host factor interactions with the HCV replicase. This new line of investigation will complement our currently funded studies of genetic, biochemical, and structural interactions between viral proteins, and accelerate progress towards a more complete understanding of the central multiprotein complex catalyzing HCV replication. We will heavily bias our studies towards the identification of functional interactions by performing pull-down assays of individual viral proteins from native complexes within an infected cell. To do this we will discover permissive sites for tag insertion within each HCV gene, we will optimize conditions to pull-down the viral protein and associated factors, and we will independently validate and perform mechanistic studies on each interaction. The requested funding will be used to jump-start this project by acquiring significant reagent sets and by supporting one currently unfunded postdoctoral associate, partially funding one Ph.D. student, and hiring an additional technician. The proposed expenditures will thereby further the aims of the Recovery Act while dramatically increasing our understanding of the HCV replicase complex. PUBLIC HEALTH RELEVANCE: Hepatitis C virus (HCV) is a liver-tropic human virus that currently infects approximately 130 million people worldwide. We are studying the mechanisms by which HCV replicates its RNA genome and produces new infectious particles, with the hopes of identifying interactions that can be exploited for the development of anti-viral drugs. Currently funded studies are investigating the interactions between viral proteins that are important for replication complex function;the new studies proposed here expand our research to explore the importance of cellular factors in HCV propagation.